1. Field of the Invention
The present invention generally relates to superficial zone protein-binding molecules and their uses, including therapeutic uses in the diagnosis, screening, and imaging in degenerative joint disease.
2. Background Art
Articular cartilage is a highly organized, heterogeneous, avascular, resilient, weight-bearing tissue that covers the ends of bones in diarthrodial (synovial) joints. The organizational arrangement of articular cartilage is marked by zonal differences. For example, the superficial zone of adult articular cartilage is distinctly different from the middle, deep, and calcified zones of the underlying cartilage in cellularity, morphology, matrix and macromolecular composition (which includes the presence of gene products made in different zones), macromolecular organization, and material properties. Among the metabolic differences is the synthesis of a proteoglycan, called superficial zone protein (SZP), which is synthesized and secreted by chondrocytes in the superficial zone of articular cartilage but is not synthesized or secreted by chondrocytes in the deeper zones of the tissue (Schumacher et al. (1994) Arch. Biochem. Biophys. 311:144-52).
SZP, which is homologous to human megakaryocyte stimulating factor precursor (MS F) and camptodactyly-arthropathy-coxa vara-pericarditis (CACP) protein, has an apparent molecular weight of 345 kDa and is substituted with keratan sulfate and chondroitin sulfate glycosaminoglycan chains (Schumacher et al. (1994) Arch. Biochem. Biophys. 311:144-52). Removal of the glycosaminoglycan side chains results in minimal change in molecular weight, which suggests that SZP has only small glycosaminoglycan chains on its core protein and that it is not an aggrecan metabolite (Schumacher et al. (1999), J. Orthop. Res 17:110-120). SZP contains large (76-78 repeats) and small (6-8 repeats) mucin-like O-linked oligosaccharide-rich repeat domains flanked by cysteine-rich N- and C-terminal domains (Flannery et al. (1999) Biochem. Biophys. Res. Comm. 254:535-541). The protein core contains potential sites for N-linked oligosaccharide and glycosaminoglycan attachment, and a putative heparin-binding domain (Id.). The heparin binding domain is encoded by exon 4 of MSF. Merberg et al. (1993) In: Biology of Vitronectins and Their Receptors (eds. Pressner et al.), pp. 45-53. Chondrocytes in the superficial zone and cells of the synovial lining, in vivo and in vitro, have been shown to synthesize SZP (Schumacher et al. (1999), J. Orthop. Res 17:110-120). Unlike other proteoglycan molecules, such as aggrecan, decorin, biglycan, and fibromodulin, very little SZP is retained in the matrix surrounding the chondrocytes (Schumacher et al. (1994) Arch. Biochem. Biophys. 311:144-52). The SZP proteoglycan present in synovial fluid has a lower molecular weight than SZP in the cartilage matrix, suggesting that either there are differences in glycosylation of SZP produced by synovial cells as compared to SZP produced by chondrocytes or that the proteoglycan is partially degraded in the synovial fluid (Schumacher et al. (1999), J. Orthop. Res 17: 110-120).
SZP forms a thin layer on the surface of adult bovine articular cartilage but not fetal articular cartilage (Schumacher et al. (1999), J. Orthop. Res 17:110-120). The thickness of the stained layer increases gradually near the junction of articular cartilage with synovium and the synovium also contains SZP (Schmid et al. (1994) Proceedings of the Orthopedic Research Society, p. 97-17.). This accumulation on adult articular cartilage has been hypothesized to be due to entrapment of SZP in an acellular collagenous layer at the surface of articular cartilage (Schumacher et al. (1999), J. Orthop. Res 17:110-120). The biosynthesis of SZP by chondrocytes has been shown to be upregulated by certain growth factors and cytokines, such as TGFxcex2 and IGF-1, but down regulated by others, such as IL-1 (Flannery et al. (1999) Biochem Biophys. Res. Comm. 254:535-541).
SZP is thought to play a role in normal articular cartilage and in degenerative joint conditions. Recently, molecular defects in human SZP have been identified in individuals with camptodactyl-arthropathy-coxa vara-pericarditis syndrome (CACP), a very rare condition that can be marked by a proliferation of synovial cells, severe limitations in joint range of motion, and non-inflammatory pericarditis (Marcelino et al. (1999) Nature Genetics 23:319-322). Accordingly, it is desirable to detect SZP in various mammalian species, including humans, in order to monitor modulations in SZP levels, localization, and function.
In accordance with the purpose(s) of this invention, as embodied and broadly described herein, this invention, in one aspect, relates to a polyclonal or monoclonal antibody or a fragment thereof having specific binding affinity for superficial zone protein (SZP), wherein the binding affinity of the antibody or fragment thereof for human superficial zone protein is the same or greater than the binding affinity for bovine superficial zone protein in a competitive binding assay, IA sys analysis, or BIAcore analysis. Preferably the ligand is SZP or a variant, fragment, or protein core thereof. The present invention further provides a hybidoma cell that produces the monoclonal antibody of the invention. The invention also provides an antibody reagent kit comprising the antibody or fragment thereof of the invention and reagents for detecting binding of the antibody or fragment thereof to a ligand. In one embodiment, the kit further comprises containers containing the antibody or fragment thereof of the invention and containers containing the reagents.
In another aspect, the invention relates to methods of detecting SZP and methods of diagnosing degenerative conditions, such as joint, connective tissue, and blood disorders. Specifically, provided is a method of detecting superficial zone protein in a sample, comprising contacting the sample with the antibody or fragment of the present invention, under conditions in which an antigen/antibody complex can form; and detecting the presence of the antigen/antibody complex, wherein the presence of the antigen/antibody complex indicates the presence of superficial zone protein in the sample. Preferably the sample is selected from the group consisting of body fluids, such as synovial fluid, tears, saliva, urine, serum, plasma, and bone marrow, and connective tissue and components thereof, such as synovium, tendon, tendon sheath, ligament, meniscus, intervertebral disk, pericardium, chondrocytes, and articular cartilage. The method of diagnosing a degenerative joint condition in a subject, as provided by the present invention, comprises obtaining a test sample from the subject; detecting superficial zone protein in the test sample; and comparing the amount of superficial zone protein in the test sample with an amount present in a control sample; a modulated amount of superficial zone protein in the test sample indicating the degenerative joint condition.
In yet another aspect, the invention relates to screening methods, including a method of screening for a substance that modulates levels of superficial zone protein, comprising contacting a test sample with the substance to be screened, wherein the test sample contains superficial zone protein-producing cells; contacting, under conditions in which an antigen/antibody complex can form, the superficial zone protein in the test sample with the antibody or a fragment of the invention; detecting the level of the antigen/antibody complex in the test sample; and comparing the level of the antigen/antibody complex in the test sample with the level of antigen/antibody complex in a control sample; a lower or higher level of the antigen/antibody complex in the test sample indicating a substance that modulates levels of superficial zone protein. The superficial zone protein-producing cells are selected from the group consisting of chondrocytes, synovial cells, pericardial cells, and bone marrow cells of any species. Further provided is a method of screening for a substance that reduces a degenerative condition, such as a degenerative joint condition, in a subject, comprising contacting a first test sample from the subject with the antibody or fragment thereof of the invention, under conditions in which an antigen/antibody complex can form; detecting the level of the antigen/antibody complex in the first test sample; treating the subject with the substance to be screened; contacting a second test sample from the subject with the antibody or fragment of the invention, under conditions whereby an antigen/antibody complex can form; detecting the level of the antigen/antibody complex in the second test sample; and comparing the level of the antigen/antibody complex in the first test sample with the level of antigen/antibody complex in the second test sample, a modulated level of the antigen/antibody complex in the second test sample indicating a substance that reduces the degenerative condition. Alternatively, in one embodiment, the test sample is compared to a known standard or to a control sample from a second untreated subject with degenerative disease.
The invention also relates to a method of screening for subjects who would benefit from treatment for a degenerative joint condition, comprising the steps of obtaining a test sample from each subject; detecting superficial zone protein in the test samples; and comparing the amount of superficial zone protein in the test samples with an amount present in a control sample, a modulated amount of superficial zone protein in the test sample indicating a subject that would benefit from treatment for the degenerative joint condition. Also provided a method of monitoring a subject""s response to a treatment for a degenerative joint condition, comprising contacting a first test sample from the subject to be monitored with the monoclonal antibody or fragment thereof of the invention, under conditions that allow formation of an antigen/antibody complex; detecting the level of the antigen/antibody complex in the first test sample; treating the subject; contacting a second test sample from the subject with the antibody or fragment thereof, under conditions whereby an antigen/antibody complex can form; detecting the level of the antigen/antibody complex in the second test sample; and comparing the level of the antigen/antibody complex in the first test sample with the level of antigen/antibody complex in the second test sample, a modulated level of the antigen/antibody complex in the second test sample indicating the subject""s response to the treatment.
Further related to the invention is a method of imaging an articular surface and/or synovium of a joint, comprising contacting the articular surface and/or synovium of the joint with the antibody or fragment of the invention, under conditions in which an antigen/antibody complex can form on the articular surface and/or synovium, wherein the antibody or fragment thereof is detectably tagged; visualizing the detectable tag in antigen/antibody complexes in a plurality of locations on the articular surface; the visualization of detectable tag in antigen/antibody complexes showing the articular surface of the joint.
Additional advantages of the invention will be set forth in part in the description that follows and, in part, will be obvious from the description or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.